Characterization of two novel myelin proteins [Elektronische Ressource] / Karin Schaefer

Karin Schaefer

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universtität München










Characterization of two novel
myelin proteins















Karin Schaefer
aus
Nordenham

2009

Erklärung
Diese Dissertation wurde im Sinne von §13 Abs. 3 der Promotionsordnung vom 29.
Januar 1998 von PD Dr. Winklhofer betreut.

Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.

München, am 15.12.2009


……………………………………..
(Karin Schaefer)




Dissertation eingereicht am 15.12.2009
1. Gutachter PD Dr. Konstanze Winklhofer
2. Gutachter Prof. Dr. Christian Haass
Mündliche Prüfung am 08.03.2010 Contents

1 SUMMARY ........................................................................................................................................... 1
2 INTRODUCTION ... 4
2.1 GLIAL CELLS ................................................................................................................................................... 4
2.2 EVOLUTION OF MYELIN .................................................................................................................................... 4
2.3 MYELINATING GLIA ......... 6
2.3.1 Myelin composition and structure ... 8
2.3.2 Axo-glia support ............................................................................................................................. 21
2.3.3 Metabolism of myelinating glia as risk factor for disease 21
2.3.4 Myelination in zebrafish ................................................................................................................. 23
2.4 MICROARRAY EXPRESSION SCREEN TO IDENTIFY NOVEL MYELIN SPECIFIC GENES ......................... 26
2.5 PROMOTERS DRIVING EXPRESSION IN MYELINATING GLIA ...................... 28
3 RESULTS ............................................................................................................................................. 31
3.1 ZWILLING .................... 31
3.1.1 Gene structure ............................................................................................................................... 31
3.1.2 Phylogenetic conservation of Zwilling-A and -B proteins and the zwilling locus............................ 32
3.1.3 mRNA expression ........................... 34
3.1.4 Zwilling protein .............................................................................................................................. 36
3.2 CLAUDIN K .................................................. 39
3.2.1 Claudin k gene ................................ 39
3.2.2 Claudin k protein structure ............................................................................ 39
3.2.3 Phylogeny ....................................................................... 41
3.2.4 mRNA expression ........................................................................................... 44
3.2.5 Claudin k antibodies ....................................................... 46
3.2.6 Claudin k protein expression and localization in cells and tissues during development ................ 48
3.2.7 Clein localization in cells ............................................................................................. 52
3.2.8 Morpholino knock down ................................................ 54
3.2.9 Other Claudins related to zebrafish myelin .................................................................................... 57
3.3 CLAUDIN K PROMOTER .................................................................. 60
3.3.1 Conservation of claudin promoter in teleost .................. 60
3.3.2 Tol2 mediated Gal4-UAS expression system ................................................................ 61
3.3.3 mb eGFP under the control of claudin k promoter specifically and strongly labels the processes of
myelinating glia. ........................................................................................................................................... 62
3.3.4 Claudin k promoter driving fluorescently tagged Claudin k ........................................................... 64
4 DISCUSSION ....................................................................................................................................... 68
4.1 ZWILLING ................... 68
4.2 CLAUDIN K .................................................................................................................. 71
4.3 CLAUDIN K PROMOTER .................................................................................................................................. 76
5 METHODS .......................................................................................................... 80
5.1 SEQUENCE DATA ANALYSIS ............................................................. 80
5.1.1 Database searches ......................................................................................... 80
5.1.2 Motive Searches............................. 80
5.1.3 Alignments ..................................................................................................................................... 80
5.1.4 Construction of phylogenetic trees 80
5.1.5 Synteny .......... 80
5.1.6 Percent identity plot ...................................................................................................................... 81
5.1.7 Ks/Ka value .................................... 81
5.2 MOLECULARBIOLOGAL METHODS .................... 81
5.2.1 5’-RACE .......................................................................................................... 81
5.2.2 RT-PCR ........................................... 82
5.2.3 Claudin k promoter ........................................................................................ 83
5.2.4 Fluorescently tagged Claudin k ...................................... 84
5.2.5 Agarose gel electrophoresis ........................................................................... 86
5.2.6 Restriction digests .......................................................................................................................... 86
5.2.7 Gateway cloning ............................ 86
5.2.8 Transformation of competent E.coli .............................. 87
5.2.9 Analyzing transformants by PCR.................................................................................................... 87
5.2.10 Amplification and preparation of plasmids ............... 87
5.2.11 Amplification and preparation of BAC ...................................................................................... 87
5.2.12 Measuring DNA and RNA absorbance 88
5.2.13 Sequencing ................................................................................................................................ 88
5.3 FISH HUSBANDRY AND CARE TAKING ................................................................................................................. 88
5.4 ZEBRAFISH EGG MICROINJECTIONS ................... 88
5.4.1 Antisense Morpholino oligonucleotide injections .......................................................................... 88
5.4.2 Injections for establishing transgenic lines .................... 89
5.5 STARTLE RESPONSE MEASUREMENT ................................................................................................................. 89
5.6 IN SITU HYBRIDIZATION .................................................................................................................................. 89
5.7 IMMUNOHISTOCHEMISTRY ............................. 90
5.7.1 General immunocytochemistry antibody staining procedure ........................................................ 90
5.7.2 Cryo sections .................................................................................................. 90
5.7.3 Whole mounts................................ 90 5.7.4 Teased spinal cord fibers ................................................................................................................ 91
5.7.5 Microscopy and imaging 91
5.8 MYELIN MEMBRANE PREPARATION .................. 91
5.9 MASS SPECTROMETRY ................................................................................................................................... 92
5.10 PROTEIN ANALYSIS ... 92
5.10.1 Lysis of embryos and brain ........ 92
5.10.2 Immunoprecipitation (IP) .......................................................................................................... 92
5.10.3 Western blot .............................................................. 93
6 MATERIAL ........................................................................................................... 94
6.1 ZEBRAFISH LINES .......................................... 94
6.2 PLASMIDS AND BACS .................................................................................................... 94
6.3 PRIMERS ...........................................................................